KR20190085068A - Sulfated hyaluronic acid functionalized with dopamine - Google Patents

Abstract

Amide bonds or functionalized with dopamine conjugated by a spacer having an amino group for forming an amide bond with the carboxyl group of hyaluronic acid and a carboxyl group for forming an amide bond with an amino group of dopamine, Discloses second order sulfated hyaluronic acids having 60 mol%, preferably 15 to 35 mol%, more preferably 20 to 25 mol% of carboxyl groups.

Description

Sulfated hyaluronic acid functionalized with dopamine

The present invention relates to sulphated hyaluronic acids functionalized with dopamine by amide linkages, which can be done directly or via an appropriate spacer group. The compound of the present invention forms a salt with a drug having an ionizable group having a positive charge, specifically, an antibiotic. It is another object of the present invention to its use for coating titanium endoprostheses which are implantable in the above-mentioned salt and generally biological or biological apparatus.

Hyaluronic acid (HA) is composed of alternating residues of D-glucuronic acid and N-acetyl-D-glucosamine, and has a molecular weight ranging from 50000 to 13 x 10 ⁶ Da It is a heteropolysaccharide of linear chain with molecular weight.

Hyaluronic acid is indeed localized in the human body, and it plays an important role as a mechanical support for cells of many tissues, specifically skin, tendons, muscles and cartilage. Interaction of HA with its CD44 membrane receptors and opioid receptors is also known.

O-sulfated HA derivatives in which -OH groups are esterified with sulfuric acid are known. O-sulphation can be carried out by known techniques (see, e. G. EP 702699 and EP 940410); "Degree of sulphation" means the mole of sulfate group per mole of HA dimer (DSmol); Specifically

Grade 1 sulfation is defined as DSmol ranging from 0.5 to 1.5;

Class 2 sulfation is defined as DSmol ranging from 1.5 to 2.5;

Class 3 sulfation is defined as DSmol ranging from 2.5 to 3.5.

In general, HAS is an excellent carrier for skin absorption of pharmacologically and biologically active molecules, as it traverses skin barriers easily and thus simplifies the passage of the substances involved.

It has also been found that HAS has pharmacological properties (WO2010130468; WO2010130466): it is a potent anti-inflammatory agent that performs its function by effective regulation of numerous pro-inflammatory and anti-inflammatory cytokine activities. HAS is therefore suitable for use in the treatment of diseases mediated by changes in cytokine levels (rheumatoid arthritis, asthma, systemic and skin autoimmune diseases, viral infections, atopic dermatitis, eczema, vitiligo, lymphoma, etc.).

In dopamine synthesis, the amino acid intermediate DOPA (l-3,4-dihydroxyphenylalanine) is known as a neurotransmitter and has also recently been studied as an adhesive material. "Ruth Mytilini edul-less foot protein (Mytilus edulis foot proteins "(Mefp, specifically Mefp-3 and Mefp-5), which are known to have a significant concentration of DOPA residues , A peduncle in which the edulis is attached to the surface constitutes a peduncle. An important characteristic of DOPA is catecholics; This suggests that the high concentration of catechol units plays an important role in promoting adhesion to various surfaces including glass, plastic, ceramic, and metal and metal oxide based surfaces. Although the mechanism by which the attachment takes place is not yet fully understood, it is known that attachment occurs in both an acidic medium (pH = 5) and an alkaline medium (pH = 8) when the catheter takes the form of quinones. Because the dopamine derivatives also have the same properties, DOPA and dopamine are defined and used without distinction in the scientific literature of adhesive activity.

Both HA " as is "and its sulfated forms are used in combination with other polymers in the coating of metals (typically titanium) and polymers (e.g., PU) prostheses to promote biocompatibility and haemocompatibility (EP1060204).

DOPA has also been used as an adhesive to promote bonding with other molecules (typically polymers) having metal cores (Lee et al., Adv Mat, 2008, 20, 4154-4157).

Finally, metal prostheses are coated with polymer-conjugated DOPA, which binds to antibiotics, thereby reducing the likelihood of bacterial growth. For example, Lee et al. (Bone, 2012, 50, 974-982) have disclosed that DOPA is conjugated to heparin and further functionalized with antibiotics and BMP2 to promote osseointegration of titanium dental prostheses. Since heparin can bind to antibiotics with positive charge, including the sulfate group, which makes the conjugate totally negative, heparin has been selected. However, the presence of heparin is critical because its well-known anticoagulant activity is problematic and can cause abnormal bleeding after transplantation.

It has been found that conjugates of sulfated hyaluronic acid (HAS) and dopamine can be advantageously used to adsorb biologically and / or pharmacologically active antibiotics or molecules with positive charge by electrostatic interactions. Conjugates of drugs or other active compounds with functionalized HAS and dopamine are generally used to coat biomedical articles, specifically titanium prostheses, to make them biocompatible and, in the case of titanium prostheses, , Which improves adhesion with the bone matrix to which they are implanted. The conjugates of sulfated hyaluronic acid (HAS) and dopamine disclosed herein are useful when used with modern titanium prostheses having a compact structure or with modern prostheses having porous (trabecular) bridged structures that are fully fusible to bones It has proven to be particularly effective. After implantation, the post prosthesis treated with the conjugate disclosed herein is biocompatible and due to its specific structure, it is clustered by the cells and fully adhered to the bone.

Conjugates of sulfated hyaluronic acid (HAS) and dopamine forming the subject of the present invention also play a major role in reducing possible infections resulting from implants. This latter aspect suggests that bacterial growth and subsequent biofilm formation is a major complication after the first review of the prosthesis than in the primary implantation stages of the artificial knee joint and hip joints, This is especially important because it is necessary. It is estimated that approximately 80% of infections resulting in prosthetic removal are due to the formation of bacterial biofilm.

The biofilm is encapsulated in a polysaccharide matrix and has a high bacterial density, attached to a solid biological or non-biological surface , Aureus (Staphylococcus aureus), S. Epidermidis (S. epidermidis), S. H. Molina T to be kusu (S. haemolyticus), and so on) are stored, a normally resistant to systemic treatment with antibiotics.

The sulfated hyaluronic acid used according to the present invention is a HAS having a grade of 2 (HAS2), that is, a molar ratio (DSmol) of sulfate groups per 1 mole of HA dimer in the range of 1.5 to 2.5.

The functionalization percentage of the carboxyl groups of dopamine and HAS2, either directly or through a spacer, is in the range of 2 to 60 mole% for both direct and indirect bonds; This is preferably in the range of 15 to 40 mol%, more preferably 20 to 32 mol% in the case of direct bonding, and is preferably in the range of 2 to 20 mol% in the case of indirect bonding.

The bond between dopamine and HAS2 is of the amide type and may be a direct bond (NH ₂ of COOH-dopamine of HA) as disclosed in Scheme A, or it may be an indirect bond when the spacer is inserted between dopamine and HAS, Are always coupled through amide bonds to maximize the interaction of the surface of the titanium to be functionalized with dopamine, thereby reducing the steric effect of HAS2. The spacer used is an alkyl chain having a length in the range of 5 to 10, preferably 5 or 10, methylene units (Scheme B) or an alkyl chain having the formula HOOC- (CH ₂ ) _n -O- [(CH ₂ ) ₂ -O ] _m - (CH ₂ ) ₂ -NH ₂ (Scheme C).

Therefore, the spacer has an amino group for forming an amide bond with the carboxyl group of hyaluronic acid, and a carboxyl group for forming an amide bond with the amino group of dopamine.

Suitable spacer compounds for use in the preparation of the class II sulfated hyaluronic acid of the present invention have the formula:

- HOOC- (CH ₂ ) _n -NH ₂ , wherein n is an integer from 5 to 10, preferably 5 or 10;

- HOOC - CH ₂ - (O - CH ₂ --CH ₂ ) _m --O - CH ₂ --CH ₂ --NH ₂ wherein m is 1 or 2.

The compounds are known or can be prepared by known methods.

The reaction between HAS2 and dopamine is carried out under known conditions for the formation of amide bonds, for example in the presence of a condensing agent such as carbonyldiimidazole (CDI) or diimide.

For the preparation of derivatives in which dopamine binds to sulfated hyaluronic acid via a spacer, it is preferred to first synthesize a dopamine-spacer intermediate, and then conjugate said intermediate with HAS2. Spacers with an appropriately protected amino group can be reacted with dopamine hydrochloride in the presence of conventional condensing agents and bases. The resulting intermediate, after removal of the protecting group, is reacted under the conditions described above for the formation of an amide bond with HAS2.

The starting hyaluronic acid may be derived from any known source, for example extraction from rooster combs (EP138572), fermentation or biosynthesis (from Bacillus, WO2012032154); In this particular case, a second class HAS is used which is produced from HA having a weight average MW range of 100,000 to 250,000 Da, specifically 180,000 to 230,000 Da, with a weighted average MW subsequently referred to as "MW 200 kDa ". The preparation is carried out by a known method (EP0702699; IT102015000073016) and reported in the examples.

The term "average molecular weight" (MW) as used herein means weight-average MW, which is calculated by the "intrinsic viscosity" method (Terbojevich et al., Carbohydr. Res., 1986, 363-377).

The compounds of the present invention can also be used as a drug carrier to coat an in-vitro prosthesis.

An implantable prosthesis, made predominantly of titanium-based metal and having a compact or strut structure, is obtained by spraying a solution of a compound of the invention into the prosthesis and, optionally, thereafter dissolving the antibiotic or biologically or pharmacologically active substance (BMP-2; TGF1 [beta]; IGF) or synthetic molecules (e.g., diclofenac acid form), which are known for their inhibitory effects in biofilm formation, .

Useful antibiotics include those having a positive charge, specifically Gentamicin, Daptomycin, Vancomycin, Ciprofloxacin, Meropenem, Amikacin, Tobramycin, Tobramycin, Polymyxin, Colistin and Bacitracin, preferably gentamycin, cholestin and adatomycin.

The antibiotics form salts with the class II sulfated hyaluronic acid functionalized with dopamine. The salts and the prostheses to which they are adsorbed are additional objects of the present invention.

The HAS2-dopamine compounds of the present invention have the following advantages over the prior art:

분무가능하다. 종래 기술은 "접착제" 폴리머를 포함하는 용액 중에, 때로 장기간 (시간) 동안 상기 보철물을 침지시킬 것을 필요로 하지만, 본 발명은 수술실에서 조차도 간단히 직접 분무 적용하여 평편하고 균질한 피복, 전체적인 멸균 유지, 및 상기 보철물 이식 전에 부착 및 건조 시간의 유의한 감소, 또는 심지어 배제를 보장한다;

Sprayable. The prior art requires that the prosthesis be immersed in a solution containing an "adhesive" polymer, sometimes for a prolonged period of time, but the present invention can be applied by simply spraying directly, even in the operating room to provide a flat, homogeneous coating, And a significant reduction or even exclusion of attachment and drying time prior to implanting the prosthesis;

금속 보철물에 완벽하게 부착된다;

Fully attached to a metal prosthesis;

생체적합성이 있다;

There is biocompatibility;

골기질과의 유착을 촉진하는데 필요로 하는, 보철물의 표면 거칠기 (surface roughness)를 유지한다;

Maintains the surface roughness of the prosthesis needed to promote adhesion with the bone matrix;

가장 잘 알려져 있는 기술 (베타선 또는 감마선 조사)에 의해 멸균가능하고, 멸균 후에, 그의 구조적 특성을 온전하게 유지하며 (도파민의 산화적 분해 없음), 그러므로 항생제와의 콘쥬게이트 후에, 또한 그의 생물학적 효능을 유지한다 (항박테리아 활성이 변화되지 않음). 이는, 필요할 때, 이식될 보철물을 HAS2-DOPA로 코팅하고, 멸균하고, 수술실에서 사용하기 전에 장시간 보관하고, 사용시에 항생제를 분무하며, 이러한 경우에 수술실에서 보철물 부착 및 건조 시간의 종료까지 기다릴 필요가 없다는 것을 의미하며, 이는 특히 장시간 수술의 경우에 중요하다;

It is possible to sterilize by the best known technique (beta-ray or gamma irradiation), to maintain its structural properties intact after sterilization (without oxidative degradation of dopamine), and therefore after its conjugation with antibiotics, (The antibacterial activity is not changed). This means that the prosthesis to be implanted can be coated with HAS2-DOPA, sterilized, stored for long periods of time before use in the operating room, sprayed with antibiotics during use, and in this case waiting for the prosthesis attachment in the operating room and the end of the drying time , Which is particularly important in the case of long-term surgery;

골모세포 (osteoblast) 재생을 자극한다;

Stimulates osteoblast regeneration;

양전하를 갖는 항생제 (또는 일반적으로 활성 분자)와 정전기적 상호작용에 적합한 음전하 세트를 형성한다. 이렇게 하여, 박테리아 생물막의 형성이 방지 또는 강하게 제한되기 때문에 감염의 발병은 현저하게 감소된다;

Forms a negative charge set suitable for electrostatic interaction with an antibiotic (or generally an active molecule) having a positive charge. Thus, the onset of the infection is markedly reduced because the formation of bacterial biofilm is prevented or strongly restricted;

술페이트 기를 포함하고 있어서 헤파린과 같은 효과는 거의 없고, 그러므로 비정상적인 출혈을 일으키지 않는다;

It contains sulfate groups and has little effect like heparin and therefore does not cause abnormal bleeding;

매우 효과적이고, 신속하고, 영구적인 방식으로 작용한다.

It works in a very effective, fast, and permanent way.

Figure 1: ESEM image of untreated titanium cylinder surface and corresponding XPS spectrum of highlighted area.
Figure 2: ESEM image of the titanium cylinder surface coated with the derivative of Example 3 and corresponding XPS spectrum of the highlighted area.
Figure 3: ESEM image of cylinder surface treated with the derivative of Example 3 and then washed.
4: S in the Genta azithromycin coated with, a third embodiment of the functionalized by HAS2-DOPA or exemplary HA-DOPA in Example 12. Chemistry titanium cylinder. Curve indicating growth inhibition of Aureus .
Figure 5: Antimicrobial activity of the HAS2-DOPA conjugate of Example 3 compared to the heparin-DOPA (HEPA-DOPA) conjugate of Example 11 coupled to gentamicin, expressed as CFU / mL.
Figure 6: Anticoagulant effect of HAS2-DOPA of Example 6 by comparison with HEPA-DOPA of Example 11.
Figure 7: Effect of HAS2-DOPA on osteoblast proliferation of Examples 3 and 12 by comparison with fibronectin.

Manufacturing example

The conjugate of the second-grade sulfated hyaluronic acid and dopamine (hereinafter referred to as HAS2-DOPA) is synthesized in two steps: the step of synthesis of HAS2 and the step of reaction between HAS2 and dopamine. HAS2 can in turn be prepared from HA salified with tetrabutylammonium (TBA; EP702699) or chlorinated with sodium (IT102015000073016).

Example 1 : Preparation of HAS2 from HA-TBA

HA ^- TBA ⁺ (MW 200 kDa) of 2.0 g dm (3.22 × 10 -3 mol; 1 eq) was dissolved in 200 mL of DMSO. When the dissolution was complete, 3.59 g of Pyr.SO3 (8 eq) was added. After standing overnight at room temperature, the product was precipitated with EtOH and the resulting precipitate was filtered, washed twice with EtOH and redissolved in 150 mL of deionized water; 8 mL of a saturated NaCl solution and 115 mL of DMSO were added and the pH was adjusted to 3.4 +/- 0.1 with 3 M NaOH. The product was precipitated with 440 mL of EtOH and the resulting precipitate was filtered off, washed with EtOH / H ₂ O mixture (80:20) and EtOH, and finally dried at 37 ° C. under vacuum.

Example 2 : Preparation of HAS2 from HA-Na

4.0 g dm; sikyeotgo the Na ⁺ (MW 200 kDa) dispersed in 220 mL DMSO ^{- -} HA of ^{(9.96 × 10 3 mol 1 eq} ); 3.6 mL of methanesulfonic acid (5 eq) was added and the mixture was left under stirring at room temperature for 24 hours. When dissolution is complete, and addition of SO ₃ · Pyr (8 eq) of 12.8 g. After standing overnight at room temperature the product was precipitated with EtOH and the resulting precipitate was filtered through Gooch, washed twice with EtOH and redissolved in 150 mL of deionized water; 8 mL of a saturated NaCl solution and 115 mL of DMSO were added and the pH was adjusted to 3.4 +/- 0.1 with 3 M NaOH. The product was precipitated with 440 mL of EtOH and the resulting precipitate was filtered off, washed with EtOH / H ₂ O mixture (80:20) and EtOH, and finally dried at 37 ° C. under vacuum.

Example 3 : Synthesis of HAS2-DOPA conjugates with 23% derivatization (direct binding)

A ^- was dissolved HAS2 sodium salt of ^{- (3 mol, 1 eq 3.3} × 10) in deionized water of 100 mL, 3.0 g of benzalkonium chloride (BA ⁺ Cl) carried 2.0 g dm is prepared as in Example 2 And dissolved individually in 100 mL of deionized water. Upon completion of the dissolution, the BA ⁺ Cl ^- solution was added to the HAS2 solution to obtain a precipitate which was filtered, washed with H ₂ O, EtOH and then with acetone and dried under high vacuum in a rotary evaporator. The separated precipitate was dissolved in 160 mL of DMSO; 0.267 g (0.5 eq) of CDI and 0.1 mL of methanesulfonic acid (0.5 eq) were then added. After 30 min, stirring at 40 째 C, 0.5 g (0.8 eq) of dopamine hydrochloride was added and the reaction was allowed to stir overnight at 40 째 C with slow stirring. 16 mL of saturated NaCl solution was added the next day and the product was precipitated with EtOH. The resulting precipitate was filtered and washed with 2 volumes of a mixture consisting of EtOH / H ₂ O (85:15), then EtOH, finally acetone. The resulting product was dissolved in 50 mL of deionized H ₂ O and dialyzed for 1 day in 0.05 M acetate buffer pH 5 for 3 days and H ₂ O adjusted to pH 5 by addition of 1 M HCl for 1 day / Por® dialysis membrane, cut-off = 12,000-14,000 Da).

The dialysis time was controlled to confirm disappearance of free dopamine in the dialysis membrane by GPC. Finally, the dialyzed product was frozen and freeze-dried.

Example 4 : Synthesis of HAS2-DOPA conjugates with 55% derivatization (direct binding)

The derivative was synthesized and characterized as described in Example 3, but starting with 2 g of HAS2 sodium salt, chlorinated with BA, 1.34 g (2.5 eq) of CDI and 2.5 g (4.0 eq) of dopamine hydrochloride Reed was reacted.

Example 5 : Synthesis of HAS2-DOPA conjugates with 31% derivatization (direct binding)

The derivative was synthesized and characterized as described in Example 3, but was initiated with 2 g of HAS2 sodium salt and was chlorinated with BA, and 0.801 g (1.5 eq) of CDI and 1.25 g (2.0 eq) of dopamine hydrochloride Reed was reacted.

Example 6 : Synthesis of HAS2-DOPA conjugates with 21% derivatization (direct binding)

The derivative was synthesized and characterized as described in Example 3 but started with 2 g of HAS2 sodium salt and was chlorinated with BA and treated with 0.134 g (0.25 eq) of CDI and 0.25 g (0.4 eq) of dopamine hydrochloride Reed was reacted.

Example 7 : Synthesis of HAS2-DOPA conjugates with 6% derivatization (direct binding)

The derivative was synthesized and characterized as described in Example 3, but was initiated with 2 g of HAS2 sodium salt and was chlorinated with BA to give 0.067 g (0.125 eq) of CDI and 0.25 g (0.4 eq) of dopamine hydrochloride Reed was reacted.

Example 8: 5% HAS2-CONH- ( CH 2) 10 -CONH- dopamine synthesis of conjugates having a derivatized (coupled indirectly through an alkyl spacer having 10 carbon atoms)

Indirect bonding involves two steps: synthesis of dopamine-spacer species followed by synthesis of dopamine-spacer and HAS2:

- 8.1 : dopamine-spacer synthesis: NH ₂ - (CH ₂ ) ₁₀ -CONH-dopamine

0.55 g of 11- (Boc-amino) undecanoic acid was dissolved in 10 mL of DMF and the carboxyl was dissolved in 0.56 g of EDC (N-ethyl-N '- (3- dimethylaminopropyl) carbodiimide hydrochloride ) And DMAP (0.07 g) was added with stirring at 0 < 0 > C in the presence of a tertiary base (TEA, 0.31 mL). 0.4 g of dopamine hydrochloride was added after 10 minutes and the mixture was left at RT overnight with stirring. Then 40 mL of dichloromethane and 40 mL of H ₂ O were added and the organic phase was extracted, washed with water, and dried on the rotovap. The BOC protecting group was released by adding 5 mL of the following acid mixture: TFA 15% / H ₂ O 5% / DCM 80% and the resulting mixture was left at RT for 20 min with stirring. The solvent was then evaporated and the product was dried.

- 8.2: HAS2-CONH- (CH 2) 10 -CONH-DOPA synthesis of

The derivative is however composite, and characterized as described in Example 3, was initiated by the sodium salt HAS2 of 1 g, were chlorinated with BA, 0.45 g of the obtained in Example 8.1 of CDI and 0.5 g NH ₂ - (CH ₂ ) ₁₀ -CONH-DOPA.

Example 9: 5% HAS2-CONH- ( CH 2) 5 -CONH- dopamine synthesis of conjugates having a derivatized (coupled indirectly through an alkyl spacer having a 5 carbon atom)

The dopamine-spacer was obtained using 6- (Boc-amino) caproic acid as the reagent and following the procedure described in Example 8.1.

Product HAS2-CONH- (CH ₂₎ was obtained in accordance with the procedure described in Example 8.2 to ₅ -CONH- dopamine.

Example _{10: HAS2-CONH- (CH 2} ) having a 5% derivatized _{2 -O- (CH 2) 2 -O} -CH 2 -CONH-DOPA synthesis of the conjugate (indirect coupling via a polyethylene glycol spacer)

Indirect bonding involves the following two steps: Synthesis of DOPA-spacer species Next, the synthesis of DOPA-spacer and HAS2

- DOPA-spacer synthesis: NH ₂ - (CH ₂ ) ₂ -O- (CH ₂ ) ₂ -O-CH ₂ -CONH-DOPA

0.64 g of Boc-NH- (PEG) -COOH. DCHA (9 atoms) was dissolved in 10 mL of DMF and the carboxyl was activated with 0.56 g of EDC (N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride) and DMAP g) was added with stirring at 0 < 0 > C in the presence of a tertiary base (TEA, 0.31 mL). After 10 minutes DOPA (dopamine hydrochloride) was added and allowed to stir at RT overnight. Then 40 mL of dichloromethane and 40 mL of H ₂ O were added, the organic phase was extracted, washed with water and dried on a rotary evaporator. The BOC protecting group was released by adding 5 mL of the following acid mixture: TFA 15% / H ₂ O 5% / DCM 80% and left stirring at RT for 20 minutes. The solvent was then evaporated and the product was dried. 0.5 g of product was obtained (83% yield).

_{- HAS2-CONH- (CH 2)} 2 -O- (CH 2) 2 -O-CH 2 -CONH-DOPA synthesis of

This derivative was synthesized as described above for the HAS ₂ -DOPA derivative but was initiated with 1 g of HAS2 sodium salt and was chlorinated with BA, yielding 0.45 g of CDI and 0.5 g of NH ₂ - (CH ₂ ) ₂ - _{O- (CH 2) 2 -O-} CH 2 was reacted with -CONH-DOPA.

Example 11 (comparative): Synthesis of Heparin (HEPA) -DOPA conjugate with 21% derivatization (Lee et al., Prepared according to 2012)

^{1.0 g dm (3.3 × 10 -} 3 mol, 1 eq) was dissolved in the heparin sodium (HEPA) in de-ionized water of 50 mL, 1.5 g benzalkonium of benzalkonium chloride (BA ⁺ Cl ^-) individually ride in 100 mL And dissolved in ionized water. Upon completion of the dissolution, the BA ⁺ Cl ^- solution was added to the HEPA solution to obtain a precipitate which was filtered, washed with H ₂ O, EtOH and then with acetone and dried under high vacuum in a rotary evaporator. The separated precipitate was dissolved in 80 mL of DMSO; 0.134 g (0.5 eq) of CDI and 0.05 mL of methanesulfonic acid (0.5 eq) were then added. After stirring for 30 minutes at 40 < 0 > C, 0.25 g (0.8 eq) of dopamine hydrochloride was added and the reaction was continued overnight at 40 < 0 > C with slow stirring. 8 mL of saturated NaCl solution was added the next day and the product was precipitated with EtOH. The resulting precipitate was filtered off and washed with 2 volumes of a mixture of EtOH / H ₂ O (85:15), then EtOH, finally acetone. The resulting product was dissolved in 50 mL deionized H ₂ O and dialyzed for 1 day in H ₂ O adjusted to pH 5 with 0.05 M acetone buffer pH 5 for 3 days and 1 M HCl added (Spectra / Por® dialysis membrane, cut-off = 3,000 Da).

The dialysis time was controlled to confirm disappearance of free dopamine in the dialysis membrane by GPC.

Example 12 (comparative): Synthesis of HA-DOPA conjugate with 23% derivatization

The HA-DOPA derivative was synthesized by the procedure described in Example 3, but initiated with 1.32 g (0.0033 moles) HA sodium salt 200 kDa and chlorinated with BA, yielding 0.267 g (0.5 eq) of CDI and 0.5 g eq) with dopamine hydrochloride.

Example 13 : Attachment test of HAS2-DOPA derivative

When dopamine acts as an adhesive at various pH values, its ability to coat HAS2-DOPA conjugates (prepared as described in Example 5) dissolved in PBS at two most typical pH values, 5 and 8, Tested:

- Solution A: 20 mg of HAS2-DOPA (31%) in 4 mL of 0.1 M MES pH 5.

- Solution B: 20 mg of HAS2-DOPA (31%) in 4 mL of 0.1 M PBS pH 8.

A fluorescent probe with positive charge at any pH (Sanguinarine hydrochloride) was used in this test to stimulate the interaction of the derivative with a positive charge antibiotic at physiological pH.

Solution A and solution B were uniformly sprayed in two titanium cylinders (d = 15 x h = 17 mm), washed with deionized water, dried in air, and resuspended in 0.1 M MES, 0.5 mg / ≪ / RTI > concentration of < RTI ID = 0.0 > The cylinder was placed under gentle agitation at RT overnight. The next day the cylinder was again washed with deionized water, dried under N ₂ flow, irradiated with a 360 nm UV lamp and visually inspected.

From the evaluation of the fluorescence emitted by the treated cylinder, it was deduced that at pH 8 the dopamine derivative (solution B) adheres best to the surface. This finding indicated that solution B is an ideal approach for implementing the present invention.

13.1 : Electron microscope (ESEM) analysis of titanium surface coating

This test demonstrated that the HAS2-DOPA derivative attached to the surface of the cylinder by spraying not only uniformly adhered to Ti but also maintained the surface roughness necessary for promoting osseointegration of bone matrix and prosthesis. A solution of 25 mg / mL HAS2-DOPA (23%; Example 3) in 0.1 M PBS, pH 8 was prepared, 1 ml of the solution was sprayed onto a Ti cylinder; This was air-dried and the surface was observed with ESEM and compared to the surface of the untreated cylinder (Figures 1 and 2). Photographic scanning was performed as well as qualitative XPS analysis (photoelectron X-ray spectroscopy, which detects the presence of organic material on the surface being analyzed). The treated cylinder was then washed to ensure that the attachment persisted on the surface even in the presence of contact with the flow of physiological fluid after in vivo implantation (FIG. 3).

The image of the treated surface clearly showed the presence of the material and was confirmed by XPS analysis. Moreover, the dried derivatives formed surface irregularities giving a rough texture to the surface, which is an important parameter for promoting the adhesion of the implant to the bone matrix.

After washing, the derivative residues were still visible at the surface, the derivatives interacting actively with Ti, and the adhesion persisted after washing.

This shows:

HAS2-DOPA 콘쥬게이트는, 특히 pH=8의 용액에서 제조될 때, 티타늄 표면에 완벽하게 부착됨;

The HAS2-DOPA conjugate is fully attached to the titanium surface, especially when prepared in solution at pH = 8;

HAS2-DOPA 콘쥬게이트는 또한 세척 후에 표면에 결합이 유지됨; 및

The HAS2-DOPA conjugate also retains binding to the surface after washing; And

HAS2-DOPA 콘쥬게이트는 보철물의 골유착을 촉진하는데 필요한 거친 질감을 금속 표면에 제공함.

The HAS2-DOPA conjugate provides the metal surface with the rough texture needed to promote osseointegration of the prosthesis.

Example 14: S. In vitro testing of Aureus bacterial growth inhibition (HAS2-DOPA or HA-DOPA and gentamicin-functionalized titanium cylinders)

The following were prepared:

Solution of HAS2-DOPA in 0.1 M PBS, pH = 8 (23%, Example 3);

Solution of HA-DOPA in 0.1 M PBS, pH = 8 (23%, Example 12);

Gentamicin solution (0.25 M MES, 25 mg / 5 ml in pH = 5.2);

0.1 M PBS, pH = 8 (control).

The HAS2-DOPA solution was sprayed in two titanium cylinders (d = 15 x h = 17 mm), air-dried for 15 minutes, gentamicin solution was sprayed and air-dried again for 15 minutes.

The same treatment was applied to two different titanium cylinders of the same size, to which the HA-DOPA derivative solution and then the gentamicin solution was sprayed by the procedure described above.

Finally, to two different titanium cylinders of the same size, a PBS solution followed by a gentamicin solution was sprayed by the procedure described above.

Each cylinder (total: 6) was then immersed once in MQ water for 5 seconds, air-dried for 15 minutes, and cultured in Staphylococcus aureus 600,000,000 CFU / mL inoculated culture (buffered peptone water (Buffered Peptone Water): 10.0 g / L of peptone, 5.0 g / L of sodium chloride, 3.6 g / L of anhydrous disodium phosphate, 1.5 g / L of potassium phosphate, Biokar Diagnostics). The culture was incubated at 37 ° C and 1 mL of supernatant was collected at a pre-set time (6 h, 12 h, 24 h, 48 h, 144 h) and the plate was seeded with PCA-Plate Count Agar 1: 10 (according to bacterial growth) for sterile saline solution for 5.0 g / L, yeast extract 2.5 g / L, glucose 1.0 g / L, bacteriological agar 12.0 g / ). ≪ / RTI > The CFU / mL count was then performed.

S of 600,000,000 CFU / mL. The percent inhibition of bacterial growth over time as compared to the initial inoculum of Aureus is shown in FIG.

The graph clearly illustrates the following:

HAS2-DOPA는 대조군 (PBS)보다 훨씬 더 효과적으로 작용한다; 이는 HAS2-DOPA가 경시적으로 조절되고, 일정한 방식으로 겐타마이신을 방출하는 것을 의미한다;

HAS2-DOPA works much more effectively than the control (PBS); This means that HAS2-DOPA is regulated over time and releases gentamicin in a uniform manner;

HAS2-DOPA는 HA-DOPA보다 훨씬 더 유의한 방식으로 작용한다;

HAS2-DOPA acts in a much more significant manner than HA-DOPA;

놀랍게도, 처음 24시간 내에 전체 항미생물 작용 이외에, HAS2-DOPA는 접종 후에 최대 144시간, 즉 7일 동안 장기 항박테리아 커버력을 유지하지만, 반면에 HA-DOPA는 에스. 아우레우스의 증식이 단 48시간 만에 재개되었다.

Surprisingly, in addition to full antimicrobial action within the first 24 hours, HAS2-DOPA maintains long-term antibacterial coverage for up to 144 hours, or 7 days, after inoculation, The proliferation of Aureus resumed in just 48 hours.

A similar experiment was carried out with the same concentration of affomycin, and the same activity profile was obtained.

Example 15 : Comparative test: Antimicrobial activity of HAS2-DOPA conjugate compared to heparin-DOPA (HEPA-DOPA) conjugate conjugated to gentamicin

The HAS2-DOPA conjugate was prepared as in Example 6 and the HEPA-DOPA derivative was prepared as in Example 11 (derivatization degree: 21%).

Each of the two titanium cylinders (d = 15 x h = 17 mm) was sprayed with HAS2-DOPA or HEPA-DOPA at a concentration of 20 mg / mL in PBS at pH 8 followed by 5 mg / mL of gentamicin was sprayed. Three successive immersion washings (5 seconds) were then performed in MQ water; The purpose of repeated washing is to remove all gentamycin that is not electrostatically bonded to the tested species. Es the cylinder to the next. Was inserted into a test tube having an inoculum of Aureus (600,000,000 CFU / mL); 1 mL of the supernatant was taken at a pre-set time, followed by plate seeding (PCA-Plate Count Agar: Tryptone 5.0 g / L, Yeast Extract 2.5 g / L, Glucose 1.0 g / L, Bacterial Agar 12.0 g / L (Biokar Diagnostics), in a sterile saline solution, scalarly diluted 1:10 (following bacterial growth). The CFU / mL count was then performed as described in FIG.

From the results the product was HAS2-DOPA, S when the derivative and the degree of concentration of the same. Was much more active in inhibiting Aureus proliferation and the activity lasted for at least 36 hours. This result is particularly important in view of the fact that the HEPA-DOPA derivative is gradually lost rapidly after 36 hours of its antibacterial activity, such as "control" (no antimicrobial activity).

Example 16 : Comparison of anticoagulant effect between HAS2-DOPA and HEPA-DOPA

The HAS2-DOPA conjugate of Example 6 and the HEPA-DOPA derivative of Example 11 were prepared.

Anticoagulant effects were assessed using pure heparin as standard. A kit (Hyphen BioMed, Biophen Heparin AT + Ref 221007) using a colorimetric method was used; Coagulation factor Xa, which was not complexed and inhibited by heparin or test polymer, was measured. The absorbance at 405 nm was directly proportional to the free Xa factor and therefore inversely proportional to the heparin-like activity of the tested species (Figure 6).

The anticoagulant effect of HAS2-DOPA is much lower than the anticoagulant effect of HEPA-DOPA at the same concentration; To obtain the same effect with the HAS2-DOPA system (50% reduction in Abs), approximately 50-fold higher concentrations are required. This means that the HAS2-DOPA conjugate contains a sulfated polymer just like heparin, but exhibits a lower anticoagulant effect than the known product.

The HAS2-DOPA conjugate conjugated to gentamicin or a similar antibacterial agent is applicable not only by spraying but also within a short period of time and has no substantial heparin-like effect and therefore does not cause abnormal bleeding, It is beneficial because it acts in a way that is far more effective, fast, and sustainable than water.

Example 17 : Evaluation of the effect of HAS2-DOPA and HA-DOPA on in vitro osteoblast proliferation

The following were prepared:

- aqueous solution of HAS2-DOPA (23%, Example 3) 10 mg / mL

- aqueous solution of HA-DOPA (23%, Example 12) 10 mg / mL

- aqueous solution of fibronectin (positive control) 20 μg / ml.

The circular top surface of two titanium cylinders (d = 15 x h = 17 mm) was sprayed with HAS2-DOPA solution and air-dried for 15 minutes; The same treatment was applied to two other titanium cylinders of the same size and the HA-DOPA solution was sprayed thereto according to the procedure described above. Finally, two identical titanium cylinders of the same size were sprayed with a solution of the fibronectin in water according to the procedure described above. Because fibronectin stimulates cell attachment, proliferation and migration, it has been widely used as a reference standard in in vitro cell proliferation experiments.

Each cylinder (total: 6) was then immersed once in MilliQ water for 5 seconds, air-dried for 15 minutes and inserted into the plate wells; Then 0.10 mL of osteoblast (Saos-2 cell line, derived from osteosarcoma) in the culture medium (McCOY'S 5A + 10% FBS) was attached to the surface of each cylinder with approximately 50,000 cells per cm ² of titanium. The cells were incubated in titanium for 4 hours at 37 [deg.] C (5% CO ₂ ); The medium was then added until the cylinder was immersed and incubated overnight under the same conditions.

The medium was removed the next day, and the cylinder was washed with PBS to remove unattached cells; The same volume of the same medium was then added to each cylinder with 10% Alamar Blue and the cylinder incubated at 37 ° C (5% CO ₂ ) for 24 hours. After incubation, fluorescence reading was performed (lambda excitation: 530 nm and emission: 590 nm); Fluorescence intensity is proportional to cell metabolism and is proportional to the number of living osteoblasts.

From the results (Fig. 7), when the degree of derivatization and the concentration were the same, HAS2-DOPA had very high osseointegration activity, almost the same as osseointegration activity of fibronectin, and surprisingly almost doubled the osseointegration activity of HA-DOPA to be. The values were statistically significant.

C- represents a negative control, ie, a titanium cylinder "as is" without seeding of the osteoblasts.

Example 18 : Evaluation of the stability of 23% HAS2-DOPA powder prepared as in Example 3 for beta-ray and gamma-sterilization

Three samples of 1 g of 23% HAS2-DOPA powder, as described in Example 3, were prepared. Each of the samples was treated as follows:

베타선으로 멸균처리;

Sterilized with Betaine;

감마선으로 멸균처리;

Sterilized with gamma rays;

멸균 처리 없음 (대조군).

No sterilization (control group).

The sample was then analyzed by different known methods, such as ¹ H-NMR (positron nuclear magnetic resonance spectroscopy), IR analysis (in KBr pellet), and finally dissolution in water after dissolving the powder in heavy water (D 2 O) And then analyzed by structural analysis of visible light UV analysis (UV-Vis).

From the analysis results, it was confirmed that the signals of the sterilized samples were the same and there was no difference between the signals and the signals of the unsterilized control group. Specifically, signals due to the formation of by-products (NMR and IR analysis) or UV-Vis absorption signaling by dopamine oxidation, for example, were not observed.

Therefore, HAS2-DOPA polymers were stable and suitable for beta-ray or gamma-ray sterilization.

Example 19 is functionalized by HAS2-DOPA, and sterilized by beta rays, then the S in the titanium cylinder treated with vancomycin. In vitro testing of Aureus bacterial growth inhibition

The following were prepared:

Solution of HAS2-DOPA in water (23%, Example 3), 10 mg / mL;

Vancomycin solution in water, 5 mg / ml.

One sample of titanium cylinder (d = 15 x h = 17 mm) (sample A, control) did not perform any treatment, sprayed a HAS2-DOPA solution onto a second titanium cylinder of the same dimensions (sample B) While air-dried.

All of the cylinders were sterilized by irradiation with beta rays, the vancomycin solution prepared subsequently was sprayed, and finally air-dried for 15 minutes.

All of the cylinders were then immersed in different solutions of MQ water 10 times for 5 seconds to remove any excess antibiotic present and air-dried for 15 minutes. The center of the Staphylococcus aureus suspension of sikyeotgo the (at 650 nm OD> 200 μL of 0.5) in the plate are uniformly distributed on the surface of Mueller-Hinton agar (BD Biosciences), inoculated with two titanium cylinders each agar surface of . The plate was incubated at 35 ° C for 18 hours, and then the diameter of the surface where bacterial growth was inhibited by the antibiotic was measured.

Sample B treated with HAS2-DOPA and sterilized proved to be much more effective in inhibiting bacterial growth than the control sample only subjected to sterilization.

This means that the structure or properties of HAS2-DOPA are not altered by the sterilization treatment, which remains adhered to the Ti and consequently retains its ability to bind to vancomycin and subsequently releases it.

Similar experiments were performed with vancomycin in the crosslinked titanium samples at the same concentrations of gentamicin and the same conditions and at the same concentrations to obtain the same bacterial growth inhibition profile as discussed above.

Therefore, by beta-sterilization treatment:

The attachment of HAS2-DOPA to titanium was unchanged

The structural properties of HAS2-DOPA were not changed

Therefore, the ability of HAS2-DOPA to bind antibiotics was not altered, thus achieving the desired antibacterial effect.

Claims (14)

Functionalized with dopamine which is conjugated directly via an amide bond or through a spacer having a carboxyl group for forming an amide bond with an amino group and a dopamine amino group for forming an amide bond with a hyaluronic acid carboxyl group, Grade 2 sulphated hyaluronic acid having a carboxyl group content of 60 mol% (mole%). The sulfated hyaluronic acid according to claim 1, wherein the dopamine is directly conjugated through an amide bond to 15-40%, preferably 20-32% of the sulfated hyaluronic acid carboxyl group. [2] The dopamine according to claim 1, wherein the dopamine comprises 2 to 20% of the sulfated hyaluronic acid carboxyl group, an amino group for forming an amide bond, and a conjugated dihydrogenated hyaluronic acid through a spacer having a carboxyl group for forming an amide bond with an amino group Sulfated hyaluronic acid which is gated. The sulfated hyaluronic acid of claim 3, wherein the spacer is a compound of the formula HOOC- (CH ₂ ) _n -NH ₂ , wherein n is an integer from 5 to 10, preferably 5 or 10. 4. The method of claim 3, wherein the spacer is a compound of the formula HOOC- (CH ₂ ) _n -O- [(CH ₂ ) ₂ -O] _m - (CH ₂ ) ₂ -NH ₂ , wherein n is 1 or 2, and m is 1 or 2. 6. A composition according to any one of claims 1 to 5, obtained by functionalisation of a level 2 sulfated hyaluronic acid prepared from hyaluronic acid having a weight average molecular weight of 100,000 to 250,000 Da, specifically 180,000 to 230,000 Da Sulfated Hyaluronic Acid. A salt of a sulfated hyaluronic acid according to any one of claims 1 to 6 and a drug having a positive charge. 8. The salt of claim 7, wherein the agent having a positive charge is selected from antibiotics, growth factors, and diclofenac in acid form. The method of claim 8, wherein the agent having a positive charge is selected from the group consisting of aminoglycoside antibiotics, Daptomycin, Ciprofloxacin, Meropenem, Vancomycin, Polymyxin, Colistin, ≪ / RTI > Bacitracin. 10. The salt of claim 9, wherein the aminoglycoside antibiotic is selected from Amikacin, Gentamicin and Tobramycin. The salt according to claim 7, wherein the agent having the positive charge is gentamycin, adatomycin, polyamicin or cholestin. The sulfated hyaluronic acid of claims 1 to 6 for use as a pharmaceutical carrier. The sulfated hyaluronic acid of claims 1 to 6 for use in coating biomedical articles. Titanium endoprostheses coated with the salt of claims 7-11.

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